ABSTRACT: The trachea is a geometrically simple organ that facilitates airflow between the larynx and the lungs. Its failure is lethal. Small defects may be resolved by removing the diseased tissue, but critical length defects require a tissue graft. In adult patients, a synthetic graft material provides sufficient function, but in pediatric patients, multiple surgeries are needed to replace grafts that become too small for their growing bodies. To address this, we plan to create 3D printed tracheal grafts from the extracellular matrix (ECM) of decellularized tissue with the potential to grow with the patient. Specific aim 1 is to synthesize and optimize a 3D printed, pro-cartilage hydrogel by modifying amine groups in decellularized ECM with methacrylate. The hydrogel will provide a matrix for mesenchymal stem cell-derived chondrocytes to mature and remodel, strengthening the 3D printed tissue to match the mechanical properties of native cartilage. Specific aim 2 is to synthesize and optimize a 3D printed, fibro-elastic hydrogel derived from tracheal fascia using similar methods. This hydrogel will mimic the matrix of the fascia that allows longitudinal flexibility. This hydrogel will be seeded with fibroblasts, and characterized by its tensile mechanics. Specific aim 3 is to combine the optimized hydrogels from aim 1 and aim 2 into a multi-tissue trachea construct supported by a customized bioreactor. Alternating between these bio-inks, we will construct a trachea-like, cylindrical, structure that will allow flexibility in the axial direction while resisting deformation in the radial direction. This construct will be cultured in a customized bioreactor, providing convective transport of oxygen and nutrients through the wall of the construct. The efficacy of the bioreactor will be evaluated by examining pH levels, oxygen concentration, and sterility. The tissue engineered tracheal construct will be compared to normal tracheal tissue through mechanical testing and histology. Eventually this research will lead to clinically relevant treatments for critical length tracheal defects, greatly improving the lives of patients. Furthermore, the development of 3D printed tracheae from tissue specific ECM will greatly advance the field of pulmonary tissue engineering and help progress towards the production of more complex organs for transplant.